Since ATP is a high energy compound and concerned in the energy metabolism of many biological bodies, measurement of ATP is used as an index for biological activity. Illustratively, measurement of ATP is used in various indexes such as viable cell count, metabolic changes in cells, control of food quality including ripeness and putridity of food, water quality check in the water quality purification and the like.
As a method for measuring ATP, a method of a high performance liquid chromatography using a reverse phase column has been described in Analytical Biochemistry, vol. 146, p. 118, 1985, Analytical Biochemistry, vol. 145, p. 9, 1985, and the like.
Examples of known methods for measuring ATP making use of enzyme reactions include a bioluminescence method in which a light generated by the reaction of firefly luciferase with its luminescence substrate luciferin in the presence of ATP is measured using a luminometer or the like (U.S. Pat. No. 5,905,029, JP-A-6-129988 and the like), a method in which the reaction is amplified by jointly using hexokinase and pyruvate kinase and ATP is finally measured by chemiluminescence using isoluminol (JP-A-64-23900 and the like), and a method in which nucleoside phosphorylase and xanthine oxidase are added to a solution containing an ATP-related compound, and hydrogen peroxide formed by the reaction is determined (JP-A-63-11848 and the like).
In addition, as a method for the enzymatic measurement of ADP as a degraded product of ATP, a method is known in which ADP is converted into ATP by the action of a kinase in the presence of a phosphate compound, the dephosphorylation compound formed by this reaction from the phosphate compound is oxidized by the action of a dehydrogenase in the presence of NAD, and NADH formed by this reaction is measured by the action of diaphorase (U.S. Pat. No. 4,923,796 and the like).
Also, examples of known measuring methods of ATP by a color reaction having a possibility of carrying out visual determination include a method in which an ATP degrading enzyme is allowed to act upon ATP, and molybdenum blue formed by the reaction of the thus formed phosphoric acid with molybdenum is measured (JP-A-4-360700 and the like), a method in which nicotinamide mononucleotide and adenylyl transferase are reacted in a solution containing ATP, and the thus formed NAD is determined by carrying out a coenzyme cycling reaction which uses reduction type NAD as the coenzyme with a oxidation reduction reaction system which uses NAD as the coenzyme (JP-A-59-166099 and the like), and a method in which an amplification reaction of ATP is carried out using AMP, glucose 6-phosphate, adenylate kinase and glucokinase, and the thus formed glucose is led to a color reaction (U.S. Pat. No. 6,043,047 and the like).
Separately from this, a case is known in which multiple coloration was realized in order to increase accuracy of visual determination in the color reaction using diaphorase and the like, by simultaneously using two or more tetrazolium compounds as the chromogen (Japanese Patent No. 1,782,359, JP-A-62-239054 and the like). However, nicotinamide adenine dinucleotide itself or glucose is considered as the substance to be measured, and only 1 molecule of the chromogen is changed to a pigment based on 1 molecule of the substance to be measured. Because of this, a measurable concentration range of the substance to be measured is limited to the concentration of the substance to be measured in the assay reagent solution. Since a concentration range of the substance to be measured is limited naturally by the assay reagents, it is not suited for practical use.
Regarding the method which uses a high performance liquid chromatography or a luminometer, it is basically a measuring method which uses a measuring apparatus, and the measuring apparatus is expensive in the actual field, so that concern has been directed towards a visually determinable ATP measuring reagent which does not require a measuring apparatus and has an easy determination handling ability.
Also, in the molybdenum blue-measuring method among the measuring methods of ATP by a color reaction having a possibility of carrying out visual determination, ATP is not directly measured but phosphoric acid formed by degrading ATP is measured. Because of this, it is highly possible to measure phosphoric acid and the like which are not related to the ATP derived from living bodies and the like. Phosphoric acid is also contained frequently in various synthetic products.
Also, in the determination method by carrying out a coenzyme cycling reaction which uses reduction type NAD as the coenzyme with a oxidation reduction reaction system which uses NAD as the coenzyme, ATP added to a system in which NAD is not present is converted into NAD, and its amplification is carried out by the cycling reaction of NAD and reduced type NAD. In this case, since the sample to be measured is mainly originated from organisms, contamination of NAD and reduced type NAD contained in the sample cannot be avoided, and contamination of NAD and reduced type NAD contained in the sample become large noises by causing a cycling reaction, so that a possibility of causing a large hindrance to the measurement of ATP is very high. Thus, when various possible noises are taken into consideration, it is considered that measurement of ATP by directly incorporating it in the reaction is necessary.
The method in which ATP is directly incorporated into the reaction by carrying out an amplification reaction of ATP using AMP, glucose 6-phosphate, adenylate kinase and glucokinase uses a reverse direction dephosphorylation reaction of glucose from glucose 6-phosphate, which is not the direction of the original catalytic reaction of glucokinase. This reverse reaction is small in comparison with the normal reaction. Since it is known that the reaction of ADP into ATP is hardly generated, it is expected that the amount of glucokinase used becomes great in carrying out this reaction system. Since it is generally known that glucokinase or the like enzyme which reacts with ATP is contaminated with very little amount of ATP, particularly when measurement of a trace amount of ATP is the object as in the the present invention, it can be easily considered that the contaminating ATP, though in an extremely small amount, will exert great influence on the measurement.
The invention contemplates providing an ATP measuring method and reagent, which can perform visual determination without requiring a high performance liquid chromatography, luminometer or similar expensive measuring apparatus and also can show good sensitivity by directly incorporating ATP and measuring ATP by making use of the normal reaction of glucokinase.